All organisms can detect DSBs and correct them in an efficient manner. It is important to fully understand how DSBs and other types of lesions contribute to genome instability. REPAIR OF DSBS IN YEAST. Using budding yeast, we were the first to directly characterize DSBs, their repair and genetic control over 35 years ago. Recently, we have modified systems for detection and revealed opportunities to address one of the early steps in repair, resection. Since yeast chromosomes can be displayed as individual bands according to size using pulse-field gel electrophoresis (PFGE), repair of individual chromosomes can be addressed. There was little if any restitution of full size chromosomal molecules in G1 diploid cells following ionizing radiation (IR). However, DSBs induced in G2 cells were rapidly repaired: >90% DSBs within 2 hr. Repair requires RAD50, RAD51 and RAD52. A critical early step in DSB repair and genome stability is resection of ends. While many studies with yeast have characterized resection at a unique DSB using site-specific endonucleases, it has been a challenge to address end events at random, dirty-ended DSBs. We developed a novel approach to address resection of IR induced DSBs. Circular chromosomes linearized by a single, random DSB migrate as a unique band during PFGE; however, within 10 min the band shifts to slower mobility and by 1 hr the apparent size increases 75 kb. This PFGE-shift was identical in WT, rad52 and rad51 strains. Mung bean nuclease digestion revealed the shift was due to resection and established PFGE-shift as a robust assay for detecting DSB resection. There was 1 to 2 kb resection per DSB end during repair in WT cells. In rad52 cells the resection rate was similar. However, in a rad50 mutant lacking the MRX complex, resection of radiation- and HO-induced DSBs was drastically reduced. As described in another project ES065073-21, we can apply the Pulse-shift approach to indirect DSBs generated during repair of closely opposed SSBs. Importantly, using a 2-D PFGE modification our approach has allowed us to identify for the first time resection at 1 or 2 ends of a DSB. In the absence of MRX or SAE2, there is loss of coordination of end-resection. Similar to results with a defined DSB, we found resection is a 2-step process: a) initiation of a short, single-strand 3 tail (100 bases) which is determined by the MRX complex and Sae2, and b) processive 5 degradation carried out by Exo1 and Sgs1/DNA2. In sae2 mutants, initiation is reduced dramatically, but this results in only a 2-fold reduction in rate of repair of IR-DSBs and only a modest reduction in survival. We establish a major role for Sae2 and the Mre11-nuclease in coordinated resection at dirty, radiation-induced DSBs but not clean, enzyme induced DSBs. The MRX complex is required for both types of breaks. Loss of either EXO1 or SGS1 reduces processivity of resection about 2-fold with little effect on DSB repair or survival. However, resection length is severely reduced in an exo1 sgs1 double mutant. Yet, similar to the sae2 mutant, repair of IR-DSBs is only decreased 2-fold and survival remains high, especially as compared to an MRX mutant. Thus, resection appears to be much greater than what is needed for efficient DSB repair and is not rate limiting in overall repair of IR-induced DSBs. Possibly its most important role is providing large ssDNA regions for signaling to prevent cell progression. Surprisingly, double-length linear molecules appeared in the WT and rad50 mutant within 1 hr after IR. Because the double length molecules were also found in a rad50 exo1 mutant, but not in rad52, they arise by a recombination pathway that is largely resection independent. DSBS, SINGLE-STRAND BREAKS (SSBS) AND REPAIR IN HUMAN CELLS. Guided by our yeast work, we developed a sensitive assay to measure DNA lesions/repair in human cells based on the ability to detect changes in the circular 180 kb Epstein Barr Virus using PFGE. Following IR, 50% of DSBs are repaired in 2-3 hr. We also discovered a supercoil form of EBV that provides opportunities to address induction and repair of SSBs, since a single SSB relaxes the supercoil. This is providing a far more sensitive and precise system for detecting various kinds of breaks than others currently available in human cells as well as opportunities to address SSBs and DSBs in the same sample in one PFGE lane. We found that the ratio SSBs:DSBs for low dose (<10kr) IR is 7, which is consistent with previous determination by other more indirect methods. We have applied our EBV approach to the role of poly ADP-ribose polymerase (PARP) inhibitors in repair. The mechanism by which PARP and inhibitors affect DNA repair has not been established. We directly determined the effects of PARP inhibition and PARP1 depletion on the repair of IR induced SSBs and DSBs in human lymphoblastoid cell lines. Parp1 protein was not essential for SSB repair based on shRNA inhibition of PARP1. Among ten widely used PARP inhibitors, seven had little or no effect on SSB repair. None of the PARP inhibitors affected DSB repair although a Ku inhibitor was highly effective at reducing repair of broken EBV. The remaining three, including Olaparib and Iniparib which are in clinical cancer therapy trials, strongly inhibited SSB repair. However, a decrease in PARP1 expression reversed the ability of PARP inhibitors to reduce SSB repair. Since Iniparub disrupts PARP1-DNA binding, the mechanism of inhibition does not appear to involve trapping of PARP at SSBs. RELATIONSHIP BETWEEN COHESION, DSBS AND UV-INDUCED RECOMBINATION. Ultraviolet light (UV) can provoke genome instability, partly through its ability to induce homologous recombination (HR). However, the mechanism(s) of UV-induced recombination is poorly understood. Although DSBs have been invoked, there is little evidence for their generation by UV. Alternatively, single-strand DNA lesions that stall replication forks could provoke recombination. Recent findings suggest efficient initiation of UV-induced recombination in G1 through processing of closely-spaced single-strand lesions to DSBs. However, other scenarios are possible since the recombination initiated in G1 can be completed in the following stages of the cell cycle. We developed a system that could address UV-induced recombination events that start and finish in G2 by manipulating the activity of the sister chromatid cohesion complex. We have shown that sister-chromatid cohesion suppresses UV-induced recombination events that are initiated and resolved in G2. By comparing recombination frequencies and survival between UV and ionizing radiation we concluded that a substantial portion of UV-induced recombination occurs through DSBs. This notion is supported by a direct physical observation of UV-induced DSBs that are dependent on nucleotide excision repair. However, a significant role of nonDSB intermediates in UV-induced recombination cannot be excluded.